Treatment of tuberculosis using immunomodulator compounds

ABSTRACT

Tuberculosis in an animal is treated by administration of a therapeutically effective amount of an immunomodulator of formula A. In formula (A), n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment, and X is an aromatic or heterocyclic amino acid or a derivative. The animal may be a mammal such as a human.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplications 60/256,478, filed Dec. 20, 2000 and 60/309,474, filed Aug.3, 2001.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to a method for treatingtuberculosis in animals, including mammals such as humans.

[0004] 2. Description of the Related Art

[0005] Tuberculosis is a chronic, infectious disease that is caused byinfection with tubercle bacilli belonging to the Mycobacterium genus.The infection may be asymptomatic for a period of time, but the mostcommon manifestations include chronic inflammation of the lungsresulting in fever and respiratory symptoms. If left untreated,significant morbidity and death can occur. Tuberculosis strikes peopleof all ages but is more common among the elderly and immunosuppressedindividuals. The disease can afflict animals, including humans, andlivestock such as cattle, hogs, and poultry. Mycobacterium tuberculosisis the most common cause of human tuberculosis, but a number of casesare due to Mycobacterium bovis. Animal tuberculosis in industrializedcountries is controlled with milk pasteurization; as a result, suchactions drastically reduce the incidence of disease caused by M. bovisin both cattle and humans. On the other hand, developing countries donot implement control measures consistently and pasteurization is rarelypracticed.

[0006] Methods of prevention, detection, diagnosis, and treatment havegreatly reduced both the number of people who contract the disease andthe number of people who die from it. Known therapies in the art includeisoniazid (isonicotinic acid hydrazide), rifampicin pyrazinamide (PZA),kanamycin, ethambutol, streptomycin, capreomicin, amicacin andcycloserine. Isoniazid is still a front-line therapy againsttuberculosis and modern short-course chemotherapy is initiated withthree drugs: isoniazid, rifampicin and PZA. Although tuberculosis cangenerally be controlled using extended antibiotic therapy, thistreatment is usually an insufficient method for prevention of the spreadof the disease.

[0007] Although certain chemotherapy and vaccine protocols have becomeavailable for the treatment of tuberculosis, the disease continues toclaim more lives per year than any other infectious disease. Thereremains a need in the art for improvements in the prevention andtreatment of tuberculosis.

BRIEF SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, tuberculosis in ananimal, such as a mammal, e.g., human, is treated by administration of atherapeutically effective amount of an immunomodulator of Formula A:

[0009] In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or apeptide fragment, and X is an aromatic or heterocyclic amino acid or aderivative thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As noted above, the present invention is generally related to amethod for treatment of tuberculosis in animals, such as mammalsincluding humans, through the administration of a therapeuticallyeffective amount of an immunomodulator. The immunomodulator isillustrated by Formula A.

[0011] In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or apeptide fragment, and X is an aromatic or heterocyclic amino acid or aderivative thereof, preferably where X=L-tryptophan or D-tryptophan.Appropriate derivatives of the aromatic or heterocyclic amino acids for“X” are: amides, mono- or di-(C₁-C₆) alklyl substituted amides,arylamides, and (C₁-C₆) alkyl or aryl esters. Appropriate acyl or alkylmoieties for “R” are: branched or unbranched alkyl groups of 1 to about6 carbons, acyl groups from 2 to about 10 carbon atoms, and blockinggroups such as carbobenzyloxy and t-butyloxycarbonyl. Preferably thecarbon of the CH group shown in Formula A has a stereoconfiguration,when n is 2, that is different from the stereoconfiguration of X.

[0012] Preferred embodiments utilize compounds such asγ-D-glutamyl-L-tryptophan, γ-L-glutamyl-L-tryptophan,γ-L-glutamyl-N_(in)-formyl-L-tryptophan,N-methyl-γ-L-glutamyl-L-tryptophan, N-acetyl-γ-L-glutamyl-L-tryptophan,γ-L-glutamyl-D-tryptophan, β-L-aspartyl-L-tryptophan, andβ-D-aspartyl-L-tryptophan. Particularly preferred embodiments utilizeγ-D-glutamyl-L-tryptophan, sometimes referred to as SCV-07. Thesecompounds, methods for preparing these compounds, pharmaceuticallyacceptable salts of these compounds and pharmaceutical formulationsthereof are disclosed in U.S. Pat. No. 5,916,878, incorporated herein byreference.

[0013] The Formula A compounds may be administered as dosages in therange of about 0.001-10 mg. Dosages may be administered one or moretimes per week, preferably on a daily basis, with dosages administeredone or more times per day. In preferred embodiments, the dosages areadministered by intramuscular injection, although other forms ofinjection and infusion may be utilized, and other forms ofadministration such as oral or nasal inhalation or oral ingestion may beemployed.

[0014] In preferred embodiments, the compounds of Formula A areadministered at a dosage within a range of about 0.01-1 mg, morepreferably at a dosage of about 0.1 mg.

[0015] Dosages may also be measured in micrograms per kilogram, withdosages in the range of about 0.001-10 micrograms per kilogram, morepreferably within the range of about 0.01-1 micrograms per kilogram, andmost preferably at about 0.1 micrograms per kilogram.

[0016] The method of treating tuberculosis with an immunomodulator ofFormula A may further include administration of a therapeuticallyeffective amount of an antimicrobial or bacteriostatic compoundeffective against tuberculosis. Suitable tuberculosis-treatingantimicrobial or bacteriostatic compounds include isoniazid,pyrazinamid, rifampicin, kanamycin, ethambutol, streptomycin,capreomicin, amicacin and cycloserine. These compounds may beadministered with dosages within a range of about 100-10,000 mg,preferably within the range of about 200 to 2,000 mg, more preferablywithin the range of about 400 to 1,000 mg. A compound of Formula A maybe administered concurrently and/or sequentially with administrationwith one or more of the tuberculosis-treating antimicrobial orbacteriostatic compounds, e.g., administered prior to, during, and/orsubsequent thereto.

[0017] In addition, the Formula A compounds may be used in thepreparation of a medicament for the treatment of tuberculosis.Preferably, the Formula A compounds are in a composition including apharmaceutically acceptable carrier.

[0018] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1

[0019] A patient, born in 1955 was admitted to the hospital withinfiltrative tuberculosis with destruction and dissemination in theupper lobe of the left lung. In addition to tuberculosis, the patienthad a duodenal ulcer, chronic bronchitis and multivalent allergy.Lymphopenia (18%) and increased ESR (25 mm/h) were present in the bloodcount. A decrease in the CD3+ subset was also indicated and infiltratewith a cavity and foci of dissemination were observed in the axilarsegment of the left lung. Aggressive traditional antituberculosistherapy was carried out including isoniazid at 600 mg/day intravenously,pyrazinamid at 1500 mg/day per os, rifampicin at 450 mg/day per os.

[0020] The traditional therapy was unsuccessful, and SCV-07 treatmentwas undertaken. Following a course of SCV-07 therapy at 0.1 mg daily for5 days, bacterial intoxication resolved, destructive cavities closed andthe sputum culture test was negative. CD3+ lymphocytes increased andtolerance to other drugs improved. Immune complexes in circulationdecreased close to normal (from 84 U to 69 U, normal level is 22-66 U).

EXAMPLE 2

[0021] A patient born in 1962 was admitted to the hospital withinfiltrative tuberculosis, destructive phase in the upper lobe and 6thsegment of the right lung and 4-5th segments of the left lung. Prior toSCV-07 therapy, symptoms of bacterial intoxication, dyspnea and ralesabove the damaged lobes were present. During tomographic examination,multiple destructive cavities on the infiltrative background wereobserved in the upper lobe and gross-focus dissemination in the 6thsegment of the right lung were observed as well. Large foci of lungdestruction surrounded by perifocal inflammation were found in thelingual lobe of the left lung. Furthermore, leukocytosis (11.4×10⁹/l)and increased ESR (36 mm/h) were observed in the blood count. Duringimmunological response assays, a decrease of CD4+ and CD8+ subsets andlow lymphocyte blasttransformation responses with PHA and PPD weredetected.

[0022] After two months of traditional therapy a small improvement inthe patient's condition was seen; however, the reoentgenological picturewas without significant change and immune responses remained inhibited.

[0023] SCV-07 was injected intramuscularly at 0.1 mg, once per day, for5 days with continued traditional antituberculosis therapy (isoniazid at600 mg/day intravenously, pyrazinamid at 1500 mg/day per so, rifampicinat 450 mg/day per os, kanamycin at 1000 mg/day intramuscularly). Theclinical improvement was significant. Sputum culture tests becamenegative, cavities closed and infiltrative foci resolved. Immunologicalresponses were close to normal. CD3+ lymphocytes increased from1.011×10⁹/l to 1.441×10⁹/and CD8+ numbers increased from 12% to 21%. Anormal range is approximately 26-40%. Lymphocyte functional abilitieswere also markedly elevated post SCV-07 treatment. Finally, theblasttransformation response with PHA increased from 29% to 40%. Anormal level is approximately 50%.

EXAMPLE 3

[0024] Patients with destructive lung tuberculosis, untreated previouslyor after inadequate antituberculosis therapy were treated. All patientsreceived antituberculosis therapy consisting of intravenous isoniazidand four to five other drugs and the condition of each patient wasexamined daily. Major diagnostic criteria included physical examination,chest X-ray, sputum bacterioscopy and culture, broncho alveolar lavageexamination. Also included were blood counts performed in all patientsprior to SCV-07 therapy and then once per week. Blood biochemical testsincluding blood fibrinogen, β-lipoproteins, urea, creatinine, C-reactiveprotein levels, activity of alanine and asparagine transaminases wereperformed once per month and more frequently if needed. Sputum cultureswere taken prior to therapy and one and three months after SCV-07therapy, and serologic assays including indirect hemaglutination tests.

[0025] Bilateral lung injury was found in 11 patients. Multi-segmentaldamage was observed in 11 cases. Other patients possessed mono- andbi-segmental lung lesions. In all cases, the pathological process wasdestructive. Cavities from 1 to 8 cm were observed in 17 X-ray films.Immature cavities were diagnosed in 3 cases. Multiple cavities, X-raymarkers and exudative inflammation predominated in all patients.

[0026] Patients were divided into two comparable groups: 20 patientswhich were treated with SCV-07 and 15 patients which were the control.Females slightly predominated the experimental group (55%). Patientsranged in age from 20 to 40 years of age. All patients had symptoms oftoxemia, including 4 patients with severe symptoms. Eight patients inthe experimental group possessed a productive cough.

[0027] Typically, the chest X-ray and tomograms prior to SCV-07treatment indicated an infiltrate-like shadow in the upper lobes. Thesixth segment of the right lung was surrounded by small infiltrates.Different multiple foci with periofocal inflammation were in axial andlingual segments of the left lung. Following two months of SCV-07therapy, the chest X-ray and tomogram indicated a resolution of theinfiltrate-like shadow, and foci in both lungs was detected.

[0028] SCV-07 treatment in humans increased the efficacy of theantituberculosis therapy in the experimental group. Toxemia symptomswere substantially resolved in half of the patients within a month.Moist rates resolved in 16 patients where body temperature decreased inall cases. Cavity closure after 3 months of observation was observed in25% of the patients from the experimental group as compared to only13.3% in the control group. Negative sputum culture tests were observedin 30% of SCV-07 treated patients compared to 13.3% in the control groupafter a month of observation and in 65% versus 33.3% respectively after3 months.

[0029] Hematological signs of tuberculosis activity including whiteblood cell counts and ESR were mostly diminished in the experimentalgroup. White blood cell counts of more than 10×10⁹/L were found in 6patients and a “left shift” was seen in 7 patients.

[0030] A lymphocyte level less than 18% was established in 10 cases andan ESR higher than 40 mm/h was seen in 4 patients. Eosinophil andmonocyte levels were close to normal prior to SCV-07 therapy and wereunchanged during therapy. Serum bilirubin, creatinine, urea, alanine andasparagine transaminase stabilized. Serum biochemical inflammationmarkers such as fibrinogen, β-lipoprotein, CRP decreased in one month.

[0031] Prior to SCV-07 therapy, a decrease in the parameters of cellimmunity was found in patients. A decrease of CD3+ lymphocytes wasdetected in 9 patients, a decrease in CD4+ production in 1 patient and adecrease in CD8+ production in 10 patients, all out of 14 examinedpatients.

[0032] Immunological studies included counting the percentage ofperipheral blood lymphocyte subsets such as CD3+, CD4+, CD8+, CD20+,CD25+ and CD95+ using monoclonal antibodies. The CD4/CD8 index wascalculated as well. Elevation of CD3+ and CD4+ cell levels were apparentin 7 out of 14 patients immediately after SCV-07 therapy. The CD4+/CD8+ratio also increased. These changes significantly improved in SCV-07treated patients in comparison to those patients in the control group.

[0033] Phytohemagglutinin (PHA) and purified protein derivate(PPD)-induced lymphocytes blasttransformation were also investigated.T-cell blasttransformation responses markedly increased in theexperimental group compared with those in the control group incomparison to inhibition of the T-lymphocyte blasttransformationresponse to PHA (44.1±2.4%) observed in 7 patients and an extremely lowT-cell response to PPD seen in 6 out of 8 studied cases prior totherapy.

[0034] IL-2 production after stimulation with the mitogens PHA and PPDin SCV-07 treated patients greatly increased.

EXAMPLE 4

[0035] This example illustrates the effect of immunization with SCV-07in 200 mice (Lab Animals Nursery, Rappolovo, Russia) prior to challengewith M. bovis 8.

[0036] SCV-07 was lyophilized in ampules at 0.1 mg per ampule, andstored at +4° C. SCV-07 was reconstituted with 1 ml of 0.9% sodiumchloride.

[0037] To induce infection, mice were injected with a M. bovis 8suspension (0.1 mg in 0.2 ml of physiological saline) into the taillateral vein. Twelve days later, animals possessing submiliary focusesin lungs after inoculation were randomized to one of seven groups.

[0038] Isoniazid therapy (10 mg/kg, subcutaneous) began. Animals withmultiple submiliary focuses continued in the study and those animals ingroups began treatment with SCV-07 on Day 20 and were provided with 5daily intraperitoneal (ip) injections. Animals were treated with asecond course of SCV-07, 2 days following the first treatment (Day 26)and were provided with 5 additional daily injections. Control groupsincluded mice without therapy (inoculation control) and mice onisoniazid therapy alone (therapy control).

[0039] Samples were harvested on days 4, 10, 17 and 24 after SCV-07therapy. At least 5 mice from each group were examined.

[0040] Lung damage was calculated during a macroscopic assay inaccordance with the number and severity of specific inflammation foci.Single submiliary foci were estimated at 0.5 units (U), multiplesubmiliary foci (<20) as 1.0 U, multiple submilliary foci (>20) as 1.5U, single miliary foci as 1.75 U, multiple associate submiliary andsingle miliary foci as 2.0 U, miliary foci (<10) as 2.25 U, multipleassociated miliary foci as 2.75 U, small caseous foci as 3.0 U,disseminate caseous as 4.0 U, damage to the entire lung as 5.0 U. In thecase of lung maceration by serous liquid, the index was increased by0.25 to 1.0 U, depending on the extent of damage.

[0041] Calculation of the weight index (WI) was accomplished accordingto the formula:

WI=organ weight (g) mouse weight (g)×100

[0042] Preliminary experimentation indicated that the particular strainof M. bovis 8 possessed low virulence. Confirmation of low virulenceincluded the examination of several factors: (1) the appearance ofsingle lung submiliary foci only on Day 11 and multiple foci on Day 19after inoculation, (2) no deaths until Day 34 after inoculation, and (3)the severity of infection in un-treated mice on Day 28 was similar tomice with lowly progressive tuberculosis on Day 26.

[0043] The use of SCV-07 therapy affects the progression oftuberculosis. SCV-07 treatment at all doses decreased the lung damageindex when measured 4 days after the end of 5 days of therapy (Day 28 ofinfection). Groups 5, 7, 8 and 9 (SVC-07 doses of 1, 0.1, and 0.01 ìg/kgip and 1 ìg/kg SCV-7 po) in comparison to untreated controls (Group 2)exhibited significant differences. Significant differences were alsoseen between Group 7 (0.1 ìg/kg ip) and Group 3 (isoniazid control).However, no significant difference occurred between untreated mice(Group 2) and those treated with isoniazid alone (Group 3).

[0044] Both 10 days and 17 days after the termination of treatment,differences between the control and experimental groups lackedsignificance. Only a decrease of the lung damage index in Group 7 (0.1ìg/kg ip) considerably differed from the control.

[0045] Maximum effects of SCV-07 therapy were seen 24 days after the endof therapy. Once again, favorable results were exhibited in Group 7 (0.1ìg/kg ip). Mice in this group significantly increased in body weight(30.8% versus 20.7% in isoniazid control Group 3), decreased in lungweight index (1.38±0.06 versus 1.07±0.23 in control Group 3) and inspleen weight index (1.59±0.25 versus 1.94±0.31 in Group 3). At thistime point, a significant decrease occurred in the growth of M boviscultured from spleens from Group 7 mice (200±0 CFU versus 275±14.1 CFUin Group 3).

[0046] It appears that treatment with SCV-07 therapy for 10 injections(Group 6) provided a better response than 5 injections at the same dose(Group 5). The amount of M. bovis growth in spleen culturessignificantly decreased for Group 6 (190±21.5 CFU versus 275±14.1 CFU inthe control. However, this trend did not occur in Group 5, 24 days aftertreatment.

[0047] After decapitation, blood was collected in Petri dishes andslides were prepared immediately, before blood coagulation. Air-driedslides were fixed in 96% ethanol for 10 min and stained by Giemsa stain.On each slide, 100-200 cells were counted and results were expressed asa percentage. For a leukocyte count, 10 ìl of blood was mixed with 190ìl acetic acid aqueous solution in a 96-well plate (Costar, Cambridge,Mass.), and under 100× magnification and oil immersion. A Goriaevhemocytometer determined the count.

[0048] Tuberculosis infection caused a decrease in total bloodlymphocyte and an increase in neutrophilic granulocytes as shown inGroup 2, the infected control. SCV-07 therapy induced changes in thesenumbers. Significant changes in treated animals were not observed asearly as 4 days after therapy, but 10 days after therapy, a decrease inmonocyte levels developed as seen in Groups 6, 7, 8 and 9 (SCV-07 dosesof 1 ìg/kg ip; 10 injections; 0.1 ìg/kg ip; 5 injections and 1 ìg/kg pocompared with Group 3, the isoniazid control). After 17 days of SCV-07therapy, an increase in lymphocyte numbers and a significant decrease inpercent of neutrophils developed in Groups 8 and 9 (0.01 ìg/kg ip and 1ìg/kg po). After 24 days, the differences between the Groups were not asevident.

[0049] Mice thymuses were collected aseptically, homogenized, suspendedin RPMI-1640 medium and filtered through two layers of sterile gauze.Cell suspensions were washed twice in RPMI-1640 with 2 mM L-glutamineand 80 ìl/ml gentamycin, then counted using a Goriaev hemocytometer.

[0050] No significant differences in basal thymic cell proliferationbetween SCV-07 treated (Groups 4 to 9) and control (Groups 1 to 3) miceoccurred. On the other hand, after 4 days of therapy, Con A stimulatedthymic cell proliferation exhibited numbers significantly higher forGroup 7 (SCV-07 dose of 0.1 ìg/kg ip) in comparison to Group 3(isoniazid control). Differences in other groups were not evident.Proliferation increased in all groups at later time points, butproliferation was significantly higher in SCV-07 treated groups. By day17, the proliferation response in Groups 7 and 8 (0.1 and 0.01 ìg/kg ip)showed essentially the same response as uninfected mice. By day 24,proliferation in Groups 6, 7 and 8 (1 ìg/kg, 10 injections ip, and 0.1and 0.01 ìg/kg ip) exhibited similarities to uninfected mice and highernumbers than the isoniazid control Group 3.

[0051] Spleens from mice were harvested aseptically, homogenized inRPMI-1640 medium (Biolot, Russia) and filtered through two layers ofsterile gauze. Erythrocytes were lysed in 0.83% percent ammoniumchloride solution after centrifugation of the homogenate. Spleen cellswere washed twice with RPMI-1640 with 2 mM L-glutamine and 80 ìl/mlgentamycin (Sigma, St. Louis, Mo.), then counted using a Goriaevhemocytometer.

[0052] Seventeen days after SCV-07 therapy, an increase in lymphocyteproduction and a significant decrease in the percentage of neutrophilsdeveloped in Groups 8 and 9 (0.01 ìg/kg ip and 1 ìg/kg po). After 24days of SCV-07 therapy, the differences between the groups were not asevident.

[0053] Spleen cell proliferation data is presented. Restoration of themitogen (Con A and LPS) induced response in SCV-07 treated animalsoccurs sooner than the isoniazid treated animals. After 24 days ofSCV-07 therapy, proliferative response with LPS in Group 6 (1 ìg/kg, 10injections ip) and with Con A in Groups 6 and 7 (1 ìg/kg, 10 injectionsip and 0.1 ìg/kg ip) did not differ from uninfected mice. Meanwhile, inGroup 3 the isoniazid control group, the proliferative response remainedlow.

[0054] The spleen cell proliferative response to tuberculin remained lowin all groups after 4 days of therapy, but later the proliferativeresponse of Group 6 (1 ìg/kg ip, 10 injections) was significantly higherthan the control.

[0055] Spleen cells and thymic cells were isolated as described above.In vitro, thymic cells were diluted to 10⁷/ml in RPMI-1640 with 2 mML-glutamine, 80 ìg/ml gentamycin and 4% heat-inactivated fetal calfserum (Sigma, St. Louis, Mo.). Spleen cells were diluted to 3×10⁶/ml inRPMI-1640 with 2 mM L-glutamine, 80 ìg/ml gentamycin and 20% inactivatedfetal calf serum. 0.5 ìg/ml Concanavalin A (Con A, Sigma, St. Louis,Mo.) was used to stimulate thymic cell proliferation. For spleen cellproliferation, 0.5 ìg/ml Con A, 10 ìg/ml lipopolysaccharide (Sigma, St.Louis, Mo.), and 50 ìg/ml tuberculin purified protein derivative (PPD)were utilized. The assay was carried out in 96-well plates and cellcultures were incubated for 72 hours, or 96 hours if tuberculin wasused, at 37° C. in humidified 5% CO₂, then pulsed with 5 ìl Ci/ml³H-thymidine overnight (approximately 16 h) and harvested on filtersusing a semi-automated harvester (Titertek™, Flow Laboratories, Norway).Filters were dried and proliferation calculated by ³H-thymidineincorporation by counting samples in a liquid scintillation counter (LKB1217 Racketa, Wallace, Sweden). Data was expressed in impulses perminute.

[0056] In order to study phagocytosis, peritoneal macrophages wereplated at a concentration of 10⁶ cells per Petri dish and mediaincluding 10⁷ Saccharomyces cerevisiae cells, opsonized by mice serumwas added. Results were evaluated using the Mann-Whitney test.Phagocytic activity was calculated after determination of percentage ofmacrophages involved in phagocytes. The phagocytic index was determinedby calculating the average number of yeast phagocytosed per one cell.Killing activity was resolved by the number of yeast digested bymacrophages after 1.5 h of incubation. Finally, the killing index wasdetermined as follows:${KI} = \frac{{phagocytic}\quad {index}\quad {after}\quad 1\quad {hr}}{{phagocytic}\quad {index}\quad {after}\quad 2.5\quad {hr}}$

[0057] Infection with tuberculosis leads to a decrease in peritonealphagocytic activity, phagocytic index and phagocytic killing of yeastcells. After infection, the average phagocytic activity on Day 28 was4.6% as compared to 64.2% in uninfected mice (p≦0.01). Phagocytickilling decreased 2-fold. Isoniazid therapy increased these phagocyticactivity indexes but not to the level as seen in uninfected mice. After4 days treatment, SCV-07 therapy markedly elevated phagocytic activityto 38.8% in Group 7 (SCV-07 dose of 0.1 ìg/kg ip) compared to 19.4% inGroup 3 (isoniazid control group, p≦0.05). SCV-07 treatment alsonormalized the phagocytic index and increased killing (244.4 versus196.4 in control, p≦0.05).

[0058] Ten days after SCV-07 therapy, a lesser effect of the drug wasobserved, perhaps as a result of the isoniazid therapy. Killingsignificantly increased compared to the control only in Group 5 (1ìg/kg, ip). A tendency toward decreased ingestion and killing in groupsof animals that displayed minimum efficacy of the drug toward severityof tuberculosis existed (Groups 8 and 9, 0.01 ìg/kg ip and 1 ìg/kg po).

[0059] After 17 days of SCV-07 therapy in mice, a decrease of ingestionand killing in Group 3 (isoniazid control). All SCV-07 treated miceexhibited increased phagocytic indexes more notably in Group 6 (1.0ìg/kg ip, 10 injections). The level of phagocytic activity in this groupremained the same as uninfected mice (60.4% versus 49.8% in Group 3) andphagocytic index (6.88 U versus 4.91 U, p=0.05). Both killing and thekilling index significantly increased and a trend toward increasedphagocytic function in other SCV-07 treatment groups was evident.

[0060] Phagocytosis in the isoniazid treatment group returned to thelevel seen in uninfected mice at 24 days after treatment. However, adecrease in phagocytic killing still existed (62.0 U in Group 3 versus178.0 in uninfected mice, p=0.05). Possibly, this was animmunosuppressive effect of isoniazid leading to a decrease inmacrophage killing. SCV-07 treatment, particularly in Groups 6 and 7(1.0 ìg/kg, 10 injections and 0.1 ìg/kg ip), significantly improvedmacrophage killing.

[0061] Mice peritoneal cells were harvested through peritoneal cavitywashing with a liquid consisting of Eagle medium (Biolot, Russia) with10% fetal calf serum and 10 IU/ml heparin. Counted cells and cellsuspensions were placed in 96-well plates (100 ìl/well). After plateswere incubated for 1 h at 37° C. in humidified CO₂, the supernatant wasremoved and the adherent cell monolayer washed 3 times with warm (37°C.) Eagle medium, dried, and then fixed and stained by 0.03% solution ofcrystal-violet in 30% methanol. A plate reader allowed for plates to beread at 595 nm and results were expressed as the extinction coefficient(related to cell number in the sample).

[0062] Peritoneal cell suspensions in Eagle media were centrifuged at1200 rpm. The media was decanted, cells were washed in 0.9% salinesolution and resuspended in 0.1% NBT HBSS solution at pH 7.2. Thesereactions occurred in 96-well plates. The counted cells were placed inthe wells in a volume of 100 ìl and incubated at 37° C. in humidifiedCO₂ for 1 h. After incubation, the supernatant was decanted, plates weredried at room temperature and cells were fixed in 70% methanol. In asolution of 2 M potassium hydroxide and 140 il DMSO, diformazane wasadded. Using a plate reader, plates were read at 640 nm and results wereexpressed as the extinction coefficient (related to cell number in thesample).

[0063] In RPMI-1640, spleen cells were diluted to a concentration of10⁷/ml with 10% fetal calf serum, 2 mM L-glutamine and 80 ìg/mlgentamycin. A 100 ìl cell suspension was added to each well of a 96-wellculture plate and cytokine production was induced by Con A at a finalconcentration of 2.5 ìg/ml. A control well included RPMI-1640. For 24hours, cells were incubated at 37° C. and humidified in 5% CO₂.Following incubation, 150 ìl of supernatant was removed from each welland stored at −70° C.

[0064] In order to determine the IL-2 concentration in cell culturesupernatants, the CTLL-2 IL-2 dependent cell line was used. CTLL-IL-2cells were washed twice in RPMI-1640 and counted with a Goriaevhemocytometer. Supernatants were diluted in 96-well culture plates atdilutions of 1:5, 1:15, 1:45 and 1:135 in a volume of 100 il. IL-2standard dilutions were also prepared. Cells were diluted to 2×105/ml inRPMI-1640 with 10% inactivated fetal calf serum, 2 mM L-glutamine, 80ìg/ml gentamycin. The 100 ìl suspension was added to each well of theculture plate and incubated for 48 h at 37° C. in 5% CO₂ humidity. Tenìl of 5 i Ci/ml ³H-thymidine was added to each well 16 h before the endof incubation. Following incubation, the cells were harvested on filtersthrough the use of a semi-automated harvester and samples were countedin a liquid scintillation counter.

[0065] A decrease of production of IL-2 in Con A stimulated spleen cellsafter tuberculosis infection occurred. On the other hand, an increase inIL-2 production was observed in all treated groups, at all time pointsafter therapy. As early as 4 days after SCV-07 therapy, IL-2 productionmarkedly increased in Groups 7 and 8 (SVC-07 doses of 0.1 and 0.01 ìg/kgip) as compared to Group 3 (41.6±5.6 U /ml and 39.1±2.8/ml versus16.6±2.6 U/ml, respectively). As of 24 days after SCV-07 therapy, theIL-2 level in Group 6 (1 ìg/kg ip, 10 injections) and Group 7 (0.1 ìg/kgip) did not differ from uninfected mice.

[0066] IL-4 and INF-γ assays in Con A-stimulated spleen cellsupernatants were also performed using Quantikine™ ELIZA kits R & DSystems, Minneapolis, Minn.) in accordance with kit instructions. Plateswere coated with monoclonal antibodies to either IL-4 or IFN-γ andblocked. Standards and samples were subsequently added. The cells wereincubated for 2 h and washed, following with the addition of acolorimetric substrate. After 30 minutes, the reaction was terminatedand plates were read at 450 nm using a plate reader (Multiscan™ 3550,BioRad, Japan).

[0067] Basal IFN-γ production 4 days after SCV-07 therapy failed todiffer from isoniazid control mice (Group 3). Ten days after SCV-07therapy however, a significant elevation of basal IFN-γ production inGroups 5, 6 and 7 (SCV-07 doses of 1 ìg/kg ip, 5 injections; 1 ìg/kg ip,10 injections and 0.1 ìg/kg ip) occurred. At 17 days after SCV-07therapy, this increase was observed in all mice. At the end of theexperiment maintenance of this increase in INF-γ production was seenonly in Group 6 (1 ìg/kg ip, 10 injections).

[0068] No significant changes in basal IL-4 production occurred. Con Astimulated the production of IFN-γ and IL-4. Notably, IL-4 and IFN-γproduction were changed in opposite directions. At 10 days after SCV-07therapy, INF-γ production exhibited stimulation in Groups 4, 5, 6 and 8(10 and 1 ìg/kg ip, 5 injections; 1 ìg/kg ip, 10 injections; and 0.01ìg/kg ip ip) and IL-4 production significantly decreased in Groups 5, 6and 7 (1 ìg/kg ip, 5 injections; 1 ìg/kg ip/10 injections, and 0.1 ìg/kgip).

[0069] INF-γ and IL-4 concentrations in blood serum. After 10 days ofSCV-07 therapy, IFN-γ levels increased in Groups 4, 5, 6 and 7 (10 and 1ìg/kg ip, 5 injections; 1 ìg/kg ip, 10 injections; and 0.1 ìg/kg ip)while IL-4 levels decreased in the same groups. These changes alsosimilarly occurred after 17 days of SCV-07 therapy. The concentration ofIFN-γ increased in Groups 4, 5, 6, 7 and 8 (significantly in group 7 and8, 0.1 and 0.01 ìg/kg ip) and simultaneously, IL-4 significantlydecreased in Groups 5, 6 and 7 (1 ìg/kg ip, 5 injections; 1 ìg/kg ip, 10injections; and 0.1 ìg/kg ip).

[0070] In mice, SCV-07 treatment during isoniazid tuberculosis therapyinfluences the severity of the disease and the strength of the immuneresponse. SCV-07 provided in 5 daily ip injections at a dose of 0.1ìg/kg significantly decreases both the lung weight index and the lungdamage index. M. bovis growth in spleen culture also decreases in thesemice as well as mice treated with 10 daily injections at a dose of 1.0ìg/kg and mice treated with 5 daily injections of 0.01 ìg/kg.

[0071] Improvement in Con A stimulated thymic cell proliferation is seenas early as 4 days after SCV-07 treatment. At 10 days after SCV-07therapy, prolific results are seen for proliferation stimulated by LPSor tuberculin. At 24 days after SCV-07 treatment, proliferativeresponses for both thymic and spleen cells are restored to nearly theuninfected animal responses.

[0072] SCV-07 stimulation of macrophage function occurs, but animprovement of ingesting and killing ability which had been decreased bytuberculosis infection and isoniazid therapy occurs. Production ofcytokines, another measure of the immune response, increases with thetreatment of SCV-07. Production of IL-2 by spleen cells decreases frominfection with tuberculosis; however, a significantly less reduction 4days after SCV-07 treatment occurs in certain groups. After 24 days oftreatment, production of IL-2 in other groups is restored to theuninfected animal level.

[0073] Finally, both basal and stimulated INF-γ production by boththymic and spleen cells increase after SCV-07 treatment. At certain timepoints, IL-4 production decreases in these same mice. Con A stimulatedproduction of IL-4 decreases in certain groups and serum levels of IL-4also reflect this change. These changes, increase of INF-γ and adecrease of IL-4 production, suggest that SCV-07 treatment is providinga shift from a T-helper cell response to a Th1-like immune response.

[0074] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, changes and modifications can be carried out withoutdeparting from the scope of the invention which is intended to belimited only by the scope of the appended claims.

We claim the following:
 1. A method for treating tuberculosis in ananimal, which comprises administering to an animal with tuberculosis atherapeutically effective amount of an immunomodulator of Formula A:

wherein n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment,and X is an aromatic or heterocyclic amino acid or a derivative thereof.2. The method of claim 1, wherein R is hydrogen, an acyl having 2 toabout 10 carbon atoms, or an alkyl having from 1 to about 6 carbons, andX is L-tryptophan or D-tryptophan, and wherein the carbon of the CHgroup shown in Formula A has a stereoconfiguration, when n is 2, that isdifferent from the stereoconfiguration of X.
 3. The method of claim 2wherein X is L-tryptophan.
 4. The method of claim 1, wherein saidimmunomodulator is selected from the group consisting ofγ-D-glutamyl-L-tryptophan, γ-L-glutamyl-L-tryptophan,γ-L-glutamyl-N_(in)-formyl-L-tryptophan,N-methyl-γ-L-glutamyl-L-tryptophan, N-acetyl-γ-L-glutamyl-L-tryptophan,γ-L-glutamyl-D-tryptophan, β-L-aspartyl-L-tryptophan, andβ-D-aspartyl-L-tryptophan.
 5. The method of claim 1 wherein saidimmunomodulator is γ-D-glutamyl-L-tryptophan (SCV-07).
 6. The method ofclaim 1 wherein said immunomodulator is administered at a dosage withina range of about 0.001-10 mg.
 7. The method of claim 1 wherein saidimmunomodulator is administered at a dosage within a range of about0.01-1 mg.
 8. The method of claim 5 wherein said SCV-07 is administeredat a dosage within a range of about 0.01-1 mg.
 9. The method of claim 8wherein said dosage is about 0.1 mg.
 10. The method of claim 1, furtherincluding administration of a therapeutically effective amount of anantimicrobial compound effective against tuberculosis wherein saidantimicrobial compound is selected from the group consisting ofisoniazid, pyrazinamid, rifampicin, kanamycin, ethambutol, streptomycin,capreomicin, amicacin and cycloserine.
 11. The method of claim 5 whereinsaid therapeutically effective amount of antimicrobial compound is adosage within a range of about 100-10,000 mg.
 12. The method of claim 10wherein said therapeutically effective amount of antimicrobial agent isa dosage within a range of about 200 to 2,000 mg.
 13. The method ofclaim 10 wherein said therapeutically effective amount of antimicrobialcompound is a dosage within a range of about 400 to 1,000 mg.
 14. Themethod of claim 1 wherein said animal is a human patient.
 15. A compoundfor use in treating tuberculosis comprising an immunomodulator with theFormula A:

wherein n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment,and X is an aromatic or heterocyclic amino acid or a derivative thereof.16. A compound for use in the preparation of a medicament for treatmentof tuberculosis comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of an immunomodulator of the Formula A:

wherein n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment,and X is an aromatic or heterocyclic amino acid or a derivative thereof.